The biosynthesis of cholesterol is a complex process that involves multiple enzymes and intermediates. Fetal cholesterol biosynthesis begins once the blood brain barrier is established at 12-18 weeks of human gestation and the demands for cholesterol biosynthesis remain high during the pre- and postnatal periods when neuronal development and myelination is in full swing. A number of devastating human syndromes are known that result from mutations in enzymes involved in cholesterol biosynthesis. These errors in biosynthesis lead to a perturbation of normal sterol profiles. As one example of such a condition, mutations in the enzyme that converts 7-dehydrocholesterol to cholesterol (the last step in biosynthesis) give rise to a devastating human disorder known as Smith-Lemli-Opitz syndrome (SLOS). The phenotype of SLOS includes a broad spectrum of CNS malformations and other anomalies as well as a higher than normal diagnosis for autism spectrum disorder. We suggest that human exposure to small molecules that affect cholesterol biosynthesis can have a profound effect on neuronal and glial ontogenesis. Indeed, small molecules that are identified as environmental threats are known to alter sterol profiles in a way that is characteristic of human sterol biosynthesis disorders such as SLOS. Small-molecule exposure could come from prescribed pharmaceuticals, from the environment or from dietary sources. Recent advances in our laboratories provide us with methods that are exquisitely sensitive for the measurement of levels of cholesterol precursors in cells, tissues and fluids. Our methods allow for analysis of these sterols in cell culture and small tissue punches of rodent brain slices and other organs at levels that are orders of magnitude more sensitive than existing methods of analysis. We will apply these methods to a screen of libraries of compounds that have been identified as potentially hazardous environmental agents. Our screen identifies compounds that either increase or decrease levels of the following cholesterol biosynthetic precursors: lanosterol, zymostenol, desmosterol, 7-dehydrodesmosterol, 7-dehydrocholesterol and we propose studies to include other sterols in the analysis. A typical workup of cells, tissues or biological fluids is a half an hour derivatization at room temperature and a one minute HPLC-MS run.